We have used the patch clamp technique combined with simultaneous meas
urement of intracellular Ca2+ to record ionic currents activated by de
pletion of intracellular Ca2+-stores in endothelial cells from human u
mbilical veins. Two protocols were used to release Ca2+ from intracell
ular stores, i.e. loading of the cells via the patch pipette with Ins(
1,4,5)P-3, and extracellular application of thapsigargin. Ins(1,4,5)P-
3 (10 mu M) evoked a transient increase in [Ca2+](i) in cells exposed
to Ca2+-free extracellular solutions. A subsequent reapplication of ex
tracellular Ca2+ induced an elevation of [Ca2+](i). These changes in [
Ca2+](i) were very reproducible. The concomitant membrane currents wer
e neither correlated in time nor in size with the changes in [Ca2+](i)
. Similar changes in [Ca2+](i) and membrane currents were observed if
the Ca2+-stores were depleted with thapsigargin. Activation of these c
urrents was prevented and holding currents at -40 mV were small if sto
re depletion was induced in the presence of 50 mu M NPPB. This identif
ies the large currents, which are activated as a consequence of store-
depletion, as mechanically activated Cl- currents, which have been des
cribed previously [1,2]. Loading the cells with Ins(1,4,5)P-3 together
with 10 mM BAPTA induced only a very short lasting Ca2+ transient, wh
ich was not accompanied by activation of a detectable current, even in
a 10 mM Ca2+-containing extracellular solution. Also thapsigargin doe
s not activate any membrane current if the pipette solution contains 1
0 mM BAPTA (ruptured patches). The contribution of Ca2+-influx to the
membrane current during reapplication of 10 mM extracellular calcium t
o thapsigargin-pretreated cells was estimated from the first time deri
vative of the corresponding Ca2+ transients at different holding poten
tials. These current values showed strong inward rectification, with a
maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8: membrane capaci
tance 59 +/- 9 pF). We conclude that store depletion in endothelial ce
lls may activate an extremely small Ca2+ current, that is consistent w
ith CRAC currents described in mast cells [3]. However, a non-electrog
enic component and/or Ca2+-entry through a non-selective pathway canno
t be ruled out.